In healthcare, there is a trend towards the development of so-called Point of Care (POC) devices, which are small devices, often with disposable components such as cartridges, that can be used in diagnosis and treatment of patients as an alternative to large and expensive analysis equipment.
A widely used diagnostic test is a Full blood count (FBC) test, which is a diagnostic test that is used to measure cellular composition of blood. It may give information about the status of an immune system of a patient, about the ability of the blood to disseminate oxygen and/or about the ability of the blood to effectively clot. As such, it is a fundamental test that is often used as an initial “general purpose” diagnostic tool or as a more targeted monitoring solution. Examples of care cycles that include a full blood count as a monitoring tool include oncology, arthritis and Crohn's disease. As many as 300 million FBC tests are performed each year in the developed world. FBC tests may further be used in chemotherapy monitoring, veterinary blood analysis applications and so on.
Currently, large scale commercial laboratory instruments known as hematology analyzers are used to automatically perform all measurements that comprise the FBC. The high cost and complexity of these devices, coupled to the need for venous blood, means that they are mostly large scale, centralized facilities. There is a clear clinical need for performing FBC in a near patient setting, particularly for applications that require a full blood count to monitor the progression and/or treatment of a disease.
Previously, microfluidic point of care devices have been developed which are capable of measuring individual components of the FBC. In that area, Hb measuring devices, WBC counters capable of performing a white blood cell differential and platelet count devices, devices which optically count and determine size of red blood cells are available. For cell counting, current hematology analyzers typically employ electrical coulter counting and/or optical scattering methods to count and differentiate white cells and to count and determine size of the red blood cells and platelets.
At the moment only few examples of micro fluidic coulter counter technologies exist. One example combines a coulter counter with a Hb measurement. Another example of counting cells is by flow-through impedance spectroscopy. This is a flow cytometry analysis which is especially suited for a micro fluidic format. This technique is capable of differentiating between lymphocytes, monocytes and neutrophils in lysed blood, and of counting and sizing red blood cells and platelets.
The current “gold-standard” for Hb measurement is the photometric cyanmethaemoglobin (HbCN) method disclosed in Standardization of hemoglobinometry II, The hemiglobincyanide method, Clin Chim Acta, 1961, 6, p. 38-44. This method involves chemical lysis of the red blood cells and subsequent labelling of all the Hb that these cells release with a cyanide ion. The labels produce a defined absorption profile with a maximum at 540 nm. By measuring the optical absorption at 540 nm, the concentration of Hb can be determined. Furthermore, the high stability of HbCN means that it is easy to supply a calibration standard.
The most common red blood cell lysis/cyanide conversion reagent is known as Drabkin's reagent. Drabkin's reagent contains Potassium Cyanide, which is extremely toxic. This reagent only works for very large dilutions in whole blood (1:251), since red blood cell lysis relies on the low ionic strength of the reagent to induce osmotic shock. This large dilution causes an inherent imprecision in the method. Furthermore, to measure the optical absorption at 540 nm, very long optical path lengths of ˜1 cm are required. Finally, in some pathological samples, turbidity can lead to erroneously high absorption readings, which in turn will give rise to an incorrect Hb concentration.
To avoid the problems associated with toxicity and turbidity, many other optical means of measuring Hb have been developed. A known point of care device uses sodium azide to convert the Hb to an azide-coordinated Hb derivative (azidemethemoglobin, HbN3). This method itself lends to short path length (0.1 mm) absorption spectroscopy, since dry reagents remove the need for dilution of the whole blood. Two absorbance readings are taken to determine the HbN3 concentration, i.e. one at the absorption maximum (565 nm) and one at 800 nm to correct for turbidity.
For the point of care WBC/Hb counter, a RBC lysis solution has been developed that preserves the WBCs while at the same time labeling the Hb molecule with imidazole. In a similar way as described above, the optical absorption of the imidazole labeled Hb species is measured at two wavelengths, i.e. one at the absorption peak and one to correct for turbidity and scattering effects for the white blood cells. The same solution may also be passed through a coulter counter to perform the cell count.
Another known lysis/Hb conversion reagent is based on sodium lauryl sulphate/sodium dodecyl sulphate (SLS/SDS). The SDS lyses all the blood cells and labels the Hb to get an SDS-coordinated derivative. Since SDS is a surfactant molecule, turbidity correction is not necessary and so a single absorption reading at 535 nm is taken to determine the Hb concentration. This method is designed for high dilutions of Hb, so the inherent imprecision present in the HbCN measurement is still present in the HbSDS one.
All the above described devices and techniques are capable of performing specific measurements from a finger-prick of blood. However, none of the above described devices and techniques are capable of measuring all parameters that are required for an FBC in a single POC measurement. Recently, a microfluidic device capable of performing a FBC in a single POC measurement has been disclosed in WO 2010/086786. This microfluidic device comprises a two sample preparation stages, one for diluting a portion of a blood sample with a lysis agent for a white blood cell count and a quench solution and providing the diluted portion to an impedance measurement means and a second dilution stage for diluting a further portion of the blood sample with a diluent for hemoglobin measurement and providing the diluted further portion to a measurement means for determining properties of red blood cells, such as RBC count, HB count and platelet count. The diluent is fed to the blood sample several times (i.e. at different points in the microfluidic network) to obtain a high dilution ratio. Consequently, only a fraction of the RBC count sample is used for the actual RBC count, with well over 90% of the various dilution stages being fed to waste.
In resistive microfluidic networks such as present in the microfluidic device disclosed in WO 2010/086786, dilution of the sample is achieved by providing a branch between a microfluidic channel transporting the sample and a microfluidic channel transporting a diluent, such as a lysing agent or a quenching solution. Consequently, a Y-shaped dilution stage is obtained having a main channel (the diluent channel) and a secondary channel branching into the main channel (the branch off the sample channel). Such Y-shaped branches are also used to branch off a small fraction of a diluted sample for further dilution, whilst the bulk of the diluted sample is fed to waste.
In order to achieve the required dilution ratio, it is possible in theory that the dimensions of the respective microfluidic channels are tuned to reduce the volume passing through the channel during a given time period. However, for most manufacturing processes, it is not practically feasible to reduce the dimensions of such a channel to below certain limits dictated by the manufacturing process. In such situations, to achieve the branching off of the correct amount of fluid, the flow rate through the branch has to be reduced, e.g. by tuning the fluidic resistance of the branch.
However, a problem associated with such low flow rates is that upon start-up of a fluidic flow through the resistive microfluidic network, bubbles can get trapped in those parts of the network that exhibit the low flow rate such as the aforementioned branch. This can significantly extend the duration of the initialization of the microfluidic device as such bubbles must be cleared from the device before the device is ready for use.